Yasuhiro Kawanishi

A novel fluor in insoluble collagen: a crosslinking moiety in collagen molecule.

A novel fluorescent compound, which was eluted slightly slower than pyridinoline on an amino acid analyzer, was obtained from acid-hydrolysate of insoluble collagen in bovine femur. Its uv absorption- and fluorescence spectra were very similar to those of pyridinoline. The results of extensive investigations of the nmr spectra in aliphatic region lead to the conclusion that it is a pyridinium derivative, tentatively named deoxypyridinoline which is very similar to pyridinoline and lacks the aliphatic hydroxyl group.

Fluorometric determination of selenium in nanogram amounts in biological materials using 2,3-diaminonaphthalene.

Abstract Nanogram amounts of selenium in biological material were determined fluorometrically using 2,3-diaminonaphthalene after digestion with nitric-perchloric acids. Linear relationship between fluorescence and selenium concentration held within 100 ng of selenium, and the experimental scattering was less than 4% for a range from 5 to 25 ng of selenium. A recovery of 99 ± 5% was achieved for 2.5 ng of selenium preadded to tuna muscle extract. The method is characterized by simple procedures and is suited for small quantities of samples.

Quantitative measurements of the interaction between monosialoganglioside monolayers and wheat germ agglutinin (WGA) by a quartz-crystal microbalance

Monosialogangliosides (GM1, GM2, GM3 and GM4) were reconstituted in lipid monolayers at the air-water interface. The binding amounts and the initial binding rates of wheat germ agglutinin (WGA) to the monosialoganglioside monolayers were quantitatively studied by use of a quartz-crystal microbalance (QCM). A QCM was horizontally attached to the monolayer from the air phase, and the binding behavior (mass increase) was followed by the frequency decrease of the QCM. WGA binding affinities for the ganglioside monolayers were influenced by hydrophilic head groups of lipid matrices, densities of gangliosides, and sequences of oligosaccharide in gangliosides. Binding of WGA to the gangliosides reconstituted in a phosphatidylcholine (sphingomyelin and distearoylphosphatidylcholine) matrix was strongly suppressed, but not in a neutral glycolipids (GlcCer, GalCer, and LacCer), dipalmitoylphosphatidylethanolamine, and dipalmitoylphosphatidylethanolamine matrix. WGA showed high affinity for monolayers containing 20 mol% gangliosides, but only low affinity for 100% ganglioside monolayers. WGA preferably binds to gangliosides in the following sequence: GM3 > GM4 >> GM2 = GM1. No affinities of WGA for GM2 and GM1 were observed. The combined techniques of monolayer and QCM have the advantages of investigating recognition properties of gangliosides.

Collagen crosslinks: Direct quantitative determination of stable structural crosslinks in bone and dentin collagens

Abstract Direct determination of the stable keto-imine forms of [3H]NaBH4-reducible precursors of dihydroxylysinonorleucine [(OH)2Lys-norLeu] and hydroxylysinonorleucine [(OH)Lys-norLeu] in bone and dentin collagen are described. Acidic thermal conditions were used to break labile imminium bonds and acetylate the resulting amino groups to prevent reformation of the original cross-links under the conditions used for NaBH4-reduction.

Pyridinoline is a real moiety of collagen

Abstract The presence of pyridinoline in collagen molecule was evidence fluorometrically. From pepsin-solubilized collagen of bovine femur bone, a native type-and SLS fibers were reconstituted, washed, and resolubilized. The solutions, which contain only collagen molecules, emitted the pyridinoline-specific fluorescence.

Naturally occurring ganglioside lactones in Minke whale brain.

Ganglioside lactones in Minke whale brain were analysed. In order to exclude the possibility of the artificial lactonization of gangliosides during the analytical procedures, lactone species were separated from their parent gangliosides at an early stage by DEAE-Sephadex A-25 column chromatography using a stepwise elution. Applying the improved method, GM41 lactones were found in grey and white matters of cerebrum and cerebellum, while GD3 monolactone was found only in cerebellar gray matter. The concentration of each ganglioside lactone was one thousandth of the parent ganglioside.

Analysis of oligo- and poly-N-acetylneuraminic acids and their lactones by capillary electrophoresis

Abstract The present work establishes the separation procedure from fragments of colominic acid, a mixture of homopolymers of N-acetylneuraminic acid (NeuAc), and their lactones by capillary electrophoresis. Using borate buffer containing sodium dodecyl sulfate and methyl cellulose, colominic acid can be separated up to triacontamer of NeuAc by capillary electrophoresis. The method can also separate lactones and enzymic degradation products of NeuAc oligomers.

Negative circular dichroism (CD) band of lactones of sialic acid polymers observed at 235 nm

Abstract NeuAc oligomers and polymers (DP2-15), isolated from a commercial colominic acid, were lactonized in 10 mM HCl or glacial acetic acid. All lactonized oligo/ polymers showed a negative circular dichroism (CD) band at 235 nm. The determination of lactonized oligo/polymer was performed by capillary electrophoresis. Molar ellipticity per lactone ring was −1.5 × 10 4 deg·cm 2 /dmol, and the molar ellipticity showed additivity with respect to the lactone ring.

An improved method for preparation of stable antigen coupled erythrocytes for passive hemolysis

Abstract The mild fixation of erythrocytes with glutaraldehyde prior to coupling to them with bis-diazotized benzidin, gave stable cells for passive hemolysis without affecting their hemolytic behavior. Furthermore, the use of proper amounts of bis-diazotized benzidin and antigen was antoher important factor in obtaining stable cells. Cells prepared in this way could be stored in phosphate buffer at low temperature, for at least 2 weeks without loss of maximal hemolytic titer and showed slight spontaneous hemolysis. The conditions described could be used with several proteins which differed in their physicochemical properties. Antigen coupled cells were also useful for immune adherence hemagglutination.

Urinary selenium levels in Japanese males and females

To elucidate the relationship between urinary selenium level and age or sex, we collected 789 single-void urine samples from inhabitants of Sagamihara area, nearby Tokyo. More than 30 samples constituted each age class of 10 year-interval for both sexes. To analyze such a large number of samples, we have developed a microfluorometri c method (Hasunuma et al. 1982). Mondragon et a1.(1971) and Yang et a1.(1983) have round remarkable differences in urinary selenium levels due fo the locality of the samples in their countries. Therefore, we examined urine samples from 9 districts throughout Japan and round a little significant differences among districts examined.